The treatment of infectious diseases caused by antibiotic resistant microorganisms requires a better understanding of how antibiotics kill susceptible cells and how resistance develops. The structural features of antimicrobial agents and their interaction with cellular targets needs to be better understood. The overall objective of this proposal is to learn how antibiotics inhibit the process of bacterial cell growth. The features of a novel antimicrobial target, inhibition of ribosomal subunit formation will be examined. This investigation will explore the features of ribosomal subunit precursors which accumulate in antibiotic-inhibited bacterial cells. Neomycin and paromomycin will be used to inhibit 30S subunit formation. Erythromycin and azithromycin will be tested for effects on 50S subunit synthesis. Antibiotic-resistant mutant strains of Escherichia coli with specific alterations in rRNA sequences will be studied. Ribosome assembly inhibition and antibiotic binding to precursors will be measured in these mutants. The mechanism of subunit assembly inhibition will also be tested by examining the components of the subunit precursor particles which accumulate in the presence of different antibiotics. Radiolabeled antibiotic binding to the assembly intermediates will be compared with binding to the subunits to determine association and dissociation rate constants and the relative equilibrium constants. The ribonucleases needed for rRNA turnover in inhibited cells will be studied. Ribosomal subunits will be reconstituted from their component rRNAs and proteins to study the process of subunit assembly inhibition in vitro. An investigation of these structures will reveal how ribosomal antibiotics can have two inhibitory activities. The findings from this work will help in assessing the effectiveness of existing antibiotics and in developing new compounds which specifically target ribosome formation in bacterial cells. PUBLIC HEALTH RELEVANCE: This is a research proposal designed to study how certain commonly used antibiotics work to kill bacterial cells. This investigation is designed to look at a new target in microbial cells, the assembly of the ribosome, an essential structure for making proteins. Learning how antibiotics stop formation of the ribosome will lead give information which can lead to the development of better antimicrobial agents, which may be more effective against antibiotic-resistant microorganisms.